Various hypotheses have been put forward to explain the aging process at both the cellular and organismal level. Among the most popular ones are accumulation of nuclear and mitochondrial DNA damage and mutations, damage to macromolecules induced by oxidative stress and other intrinsic chemical reactions, telomere erosion and dysfunction, activation of oncogenes, and a deterioration of stem cell renewal in proliferating tissues. Most likely, an overlap of all of these phenomena will contribute to cellular and organismal aging. However, here we propose a new paradigm: that alterations of the epigenome contribute substantially to the aging process. In this application, we propose that epigenetic changes are a hallmark of aging. We will analyze several epigenetic marks including DNA CpG methylation and chromatin modifications in cells that undergo aging in vitro and in vivo. In addition, we will analyze these marks in Cockayne syndrome cells, a premature aging syndrome. This epigenome mapping project will make use of the methylated CpG island recovery assay (MIRA) technique for DNA methylation analysis and chromatin immunoprecipitation for analysis of histone modifications. Genome-wide analysis will be accomplished by microarray and high throughput DNA sequence analysis. This grant proposes an in-depth analysis of the epigenome, at the level of histone modifications, DNA methylation and gene expression during the time course of aging and in premature aging syndromes. A homogenous cell type derived from identical body sites and obtained serially from the same individuals will enable us to make meaningful comparisons. The data will allow an estimation of the extent and specificity of epigenetic changes that occur during the aging process, and hence we will be in a position to better assess the exact contribution of epigenetics to aging.